Enrichment of innate immune cells from PBMC followed by triple cytokine activation for adoptive immunotherapy.

Abstract

Innate immune cells [Natural killer (NK) and gamma-delta (γδ) T-cells] have the advantage of mediating graft versus leukemia (GVL) without graft versus host disease (GVHD). Therefore, the infusion of activated innate immune cells post allogenic hematopoietic stem transplant (AHSCT) is a promising adoptive immunotherapy strategy for relapsed and/or refractory myeloid malignancies. Microbead depletion of T-cells and B-cells has been used as a graft manipulation method to prevent GVHD post haploidentical AHSCT. These grafts are enriched for NK and γδ T-cell receptor (TCR+) cells. Brief ex vivo activation of purified NK cells with interleukin (IL)-12, IL-18, and IL-15 [triple cytokines (TC)] has been shown to produce cells with a memory like function and significantly enhanced leukemia cytotoxicity. In our studies we depleted αβ TCR+ and CD19+ B-cells from healthy donors' peripheral blood mononuclear cells (PBMC) using microbeads; enriching the frequency of NK and γδ TCR+ cells. Following overnight TC incubation, we observed that these innate immune cells were activated based on phenotypic expression of CD69 and CD25. Further, we observed increased cytotoxicity of TC activated innate immune cells against NK sensitive and NK refractory leukemic cell targets. Further, the presence or absence of monocytes did not alter activation marker expression or in vitro cytotoxicity of innate immune cells. Additionally, we observed correlation between target cytotoxicity and mature activated NK phenotypes (CD56dim or CD56dim with co-expression of the activation markers CD69+ and/or CD25+). This approach of depleting T- and B-cells from PBMCs, combined with overnight TC activation, provides a novel cell population for donor lymphocyte infusion (DLI) post AHSCT.